The focus of this project in the Laboratory of Molecular Biology is to understand the mechanisms that control the birth and death of cells in the central nervous system. In the last year, stem cells have been widely discussed for their clinical potential and for the ethical concerns they raise. The work of this group has contributed to the identification and characterization of stem cells in the nervous system. The early nervous system has a complex morphology but the proliferating multipotential precursors to neurons and glia share many differentiation mechanisms. Similar multipotential stem cells are even found in the adult brain. Because there may be one class of stem cell and they can differentiate into functional neurons, astrocytes and oligodendrocytes, the potential uses of these stem cells in neurodegenerative disease has generated a great deal of excitement.Stem cells are often identified by expression of nestin, a specific intermediate filament protein. Work from our group first showed that nestin was expressed in the cells that generate neurons. We have identified sequences in the nestin gene that control expression in the developing nervous system. Two control sites have been identified. One region controls expression in the majority of cells, the stem cells that give rise to neurons and glia. The second control site is responsible for gene expression in restricted sites that occur at the junctions of the major brain regions. This discovery is exciting because the nestin gene reveals a second type of cell in the developing brain. Interactions between this second cell type and the widely analyzed stem cell may be important in specifying the distinct regions of the developing brain.The differentiation of stem cells to multiple fates is an important theme of stem cell biology. Defined extracellular factors controlling stem cell differentiation to different fates have been identified. Ligands that activate specific second messenger pathways have been identified that down regulate the nestin gene and direct stem cell differentiation to different fates. Major cell types of the brain are generated from the stem cells of the central nervous system (CNS). A striking feature of CNS stem cells is that they can generate the major cell types of the nervous system rapidly and efficiently in tissue culture in response to known signals. For example, astrocyes are generated when the stem cells are exposed to the growth factor CNTF (ciliary neurotrophic factor) and stem cells of the peripheral nervous system (PNS) are generated when the stem cells are treated with another growth factor BMP (bone morphogenetic protein). Because we are studying these events in the controlled environement of a tissue culture dish we are confident that these factors are instructing the cells to adopt new fates. Cells that would become neurons are adopting these new fates. The speed and specificity of these responses lies at the heart of the interest in stem cells.How does the stem cell decide to differentiate into one or other fate? One possible mechanism would be for the extracellular signal to be very special and to carry unique information. However, as is implied by the names of these signals they have other functions. Indeed, even their actions on stem cells are confusingly similar as well as different. For example, the BMP proteins can cause CNS stem cells to become astrocytes as well as the stem cells of the PNS. Recently, we focused on BMP actions using mouse genetics as well tissue culture methods. The different actions of BMP are achieved by a remarkably simple mechanism where two distinct second messenger systems can be activated depending on the level of action of signals regulated by CNTF. This is not the place to discuss the complexity of the biochemistry but it is clear that we are begining to understand how the stem cell decodes the extracellular signals to generate specific fates.A question of obvious interest is what kind of neurons do stem cells generate? In the first instance, it is important to show that stem cells make functional neurons. After all, if the stem cells only made some stunted cell that could not work properly we would be disappointed and cautious about promoting the potential uses of stem cell technology. To answer this question we have developed a major focus on neuronal differentiation.Neurons differentiate in the presence of non-neuronal cells. We have shown that a signal released by glial cells regulates the early steps in neuronal differentiation. The later steps in the maturation of neurons to form functional synpases have also been analyzed in tissue culture systems developed in our group. The neurotrophins are an important family of proteins that control features of neuronal differentiation. We have shown that neurotrophins can turn on synapses in hippocampal neurons. It is important to not that different neurotrophins turn on both excitatory and inhibitory synapses. The same mechanisms regulate synpase formation in neurons derived from stem cells. These results are encouraging because they suggest that at least these hippocampal stem cell can make functional neurons.Until now we have focussed on stem cells and how they give birth to the major cell types of the brain. Of course we know that cells in animals also die. Neurons are believed to die if they do not make functional synapses. This concept plays an important role in the way we think about how the brain is constructed. It is also an important part of our thinking when we explain the progressive nature of brain diseases. The aphorisms ?fire together wire together? and ?use it or loose it? capture the wide implications of this simple idea. The idea is that functional synapses cause the release of proteins that keep neurons alive.In the ?neurotrophic hypothesis?, synaptic transmission causes release of limiting amounts of proteins that maintain synapse function and promote neuronal survival. There are few examples where the mechanism linking synaptic activity, neurotrophic protein release and neuronal survival can be directly demonstrated. We have shown in dissociated hippocampal neurons that neurotrophins are released by excitatory synaptic activity. The released proteins promote synapse function and neuronal survival. We propose a model where synaptic activity regulates the balance between exciatation and inhibition through the distinct actions of different neurotrophins. This model system may be an ideal place to define the mechanisms that control the survival of neurons.There is now a maturity to the work in our group. The origin of neurons from stem cells and the survival/death of neurons in synaptic circuits can be analyzed in simple models. The importance of the applications of stem cell technology is widely recognized. The extension of this approach to the synaptically active neuron promises exciting times ahead in the laboratory and the applications of this basic knowledge.